T cell mediated immune responses are the net result of the behavior of individual cells in the T cell compartment. The use of TCR transgenic mice have enabled the identification of antigen-specific cells within a lymphocyte population, for determination of cell cycle status, activation phenotype, and viability. These studies have made important advances, but have been limited by the inability to examine effector functions such as proliferation and cytokine expression on a single cell basis. Recently, we have adapted and refined methods to track the proliferative history of individual cells within a population flow cytometry. Our studies indicate that TCR and CD28-mediated costimulation serve to cooperatively increase the frequency of cells which enter the proliferative pool, and the number of mitoses they undergo. Surprisingly, even with maximal levels of TCR plus co-stimulatory signals sufficient to activate 95% of the population, roughly 40% if T cells never divide yet remain viable. When isolated by flow cytometry and restimulated, these cells exhibit anergic behavior (refractory to restimulation but rescuable with IL-2). These studies raise a series of interesting questions which we will address. To do so we have adapted our system for use with flow cytometric detection of intracellular cytokines and cell surface detection of a transgenic TCR. Aim #1 will use in vitro studies to define the parameters which determine the frequency of cells which enter the proliferative pool, focusing on precursor percentage, T cell:APC ratio, APC type, and the availability of co-stimulatory and off signals. This aim will analyze the cell cycle status and checkpoint of non-proliferative cells. Aim #2 will determine if cytokine secretion (either qualitative or quantitative) is linked to proliferative history. Studies will be performed in vitro and using in vivo adoptive transfer techniques. Aim #3 will ask how TCR and CD28 signals influence cell survival in vitro and in vivo. These studies will also use mice transgenic (on the T cell lineage) for the CD28- inducible cell survival gene bcl-x to permit us to separately study the role(s) of the inductive effects of CD28 stimulation on cytokine and bcl-x expression. These studies should provide important insights into the control of T cell responses and assist in the development of strategies to modulate the immune system and induce tolerance. We will work with Dr. Monroe, whose project examines similar parameters during B cell responses. Dr. Greene's project will characterize biochemical events during anergy, and we will apply his findings to our system to examine the basis of primary non-responsiveness.